The human ether-a-go-go related gene (hERG) encodes the rapidly delayed inward rectifying potassium channel (IKr) that profoundly effects the repolarization of the human ventricle (see, Curran, M. E.; Splawski, I.; Timothy, K. W.; Vincent, G. M.; Green, E. D.; Keating, M. T., A molecular basis for cardiac arrhythmia: HERG mutations cause long QT syndrome. Cell 1995, 80, (5), 795-803; Sanguinetti, M. C.; Jiang, C.; Curran, M. E.; Keating, M. T., A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the IKr potassium channel. Cell 1995, 81, (2), 299-307; Trudeau, M. C.; Warmke, J. W.; Ganetzky, B.; Robertson, G. A., HERG, a human inward rectifier in the voltage-gated potassium channel family. Science 1995, 269, (5220), 92-5; and Warmke, J. W.; Ganetzky, B., A family of potassium channel genes related to eag in Drosophila and mammals. Proc Natl Acad Sci USA 1994, 91, (8), 3438-42). Block of IK, repolarizing current flowing through the channel in ventricular muscle can result in prolongation of the Q-T interval, a characteristic electrocardiogram pattern termed torsades de pointes, and potentially lethal arrhythmia (see, Sanguinetti, M. C.; Tristani-Firouzi, M., hERG potassium channels and cardiac arrhythmia. Nature 2006, 440, (7083), 463-9; and Haverkamp, W.; Breithardt, G.; Camm, A. J.; Janse, M. J.; Rosen, M. R.; Antzelevitch, C.; Escande, D.; Franz, M.; Malik, M.; Moss, A.; Shah, R., The potential for QT prolongation and proarrhythmia by non-antiarrhythmic drugs: clinical and regulatory implications. Report on a policy conference of the European Society of Cardiology. Eur Heart J 2000, 21, (15), 1216-31). The promiscuous nature of this channel, referred to herein as the hERG K+ channel, to bind a diverse set of chemical structures (see, Cavalli, A.; Poluzzi, E.; De Ponti, F.; Recanatini, M., Toward a pharmacophore for drugs inducing the long QT syndrome: insights from a CoMFA study of HERG K(+) channel blockers. J Med Chem 2002, 45, (18), 3844-53), coupled with the potential fatal outcome that may emerge from that interaction, have resulted in the recommendation from the International Congress of Harmonization and the U.S. Food and Drug Administration that all new drug candidates undergo testing in a functional patch-clamp assay using the human hERG protein, either in native form or expressed in recombinant form (see, Bode, G.; Olejniczak, K., ICH topic: the draft ICH S7B step 2: note for guidance on safety pharmacology studies for human pharmaceuticals. Fundam Clin Pharmacol 2002, 16, (2), 105-18). Although automated, high-throughput patch-clamp methods have been recently developed, such systems require specialized operators, live cells, and a substantial capital investment (see, Bridgland-Taylor, M. H.; Hargreaves, A. C.; Easter, A.; Orme, A.; Henthorn, D. C.; Ding, M.; Davis, A. M.; Small, B. G.; Heapy, C. G.; Abi-Gerges, N.; Persson, F.; Jacobson, I.; Sullivan, M.; Albertson, N.; Hammond, T. G.; Sullivan, E.; Valentin, J. P.; Pollard, C. E., Optimisation and validation of a medium-throughput electrophysiology-based hERG assay using IonWorks HT. J Pharmacol Toxicol Methods 2006, 54, (2), 189-99; and Dubin, A. E.; Nasser, N.; Rohrbacher, J.; Hermans, A. N.; Marrannes, R.; Grantham, C.; Van Rossem, K.; Cik, M.; Chaplan, S. R.; Gallacher, D.; Xu, J.; Guia, A.; Byrne, N. G.; Mathes, C., Identifying modulators of hERG channel activity using the PatchXpress planar patch clamp. J Biomol Screen 2005, 10, (2), 168-81). Further, since patch-clamp testing is costly, and because numerous, chemically-diverse scaffolds block the hERG K+ channel, strategies to mitigate potential cardiac liability during early-stage drug development typically employ a binding assay to predict the ability of a compound to block hERG current in the functional patch-clamp assay (see, Whitebread, S.; Hamon, J.; Bojanic, D.; Urban, L., Keynote review: in vitro safety pharmacology profiling: an essential tool for successful drug development. Drug Discov Today 2005, 10, (21), 1421-33; and Diaz, G. J.; Daniell, K.; Leitza, S. T.; Martin, R. L.; Su, Z.; McDermott, J. S.; Cox, B. F.; Gintant, G. A., The [3H]dofetilide binding assay is a predictive screening tool for hERG blockade and proarrhythmia: Comparison of intact cell and membrane preparations and effects of altering [K+]o. J Pharmacol Toxicol Methods 2004, 50, (3), 187-99).
Radioligand binding assays that use [3H]-dofetilide (see, Diaz, G. J.; Daniell, K.; Leitza, S. T.; Martin, R. L.; Su, Z.; McDermott, J. S.; Cox, B. F.; Gintant, G. A., The [3H]dofetilide binding assay is a predictive screening tool for hERG blockade and proarrhythmia: Comparison of intact cell and membrane preparations and effects of altering [K+]o. J Pharmacol Toxicol Methods 2004, 50, (3), 187-99; and Finlayson, K.; Turnbull, L.; January, C. T.; Sharkey, J.; Kelly, J. S., [3H]dofetilide binding to HERG transfected membranes: a potential high throughput preclinical screen. Eur J Pharmacol 2001, 430, (1), 147-8), [3H]-astemizole (see, Chiu, P. J.; Marcoe, K. F.; Bounds, S. E.; Lin, C. H.; Feng, J. J.; Lin, A.; Cheng, F. C.; Crumb, W. J.; Mitchell, R., Validation of a [3H]astemizole binding assay in HEK293 cells expressing HERG K+ channels. J Pharmacol Sci 2004, 95, (3), 311-9), or [35S]-MK499 (see, Wang, J.; Della Penna, K.; Wang, H.; Karczewski, J.; Connolly, T. M.; Koblan, K. S.; Bennett, P. B.; Salata, J. J., Functional and pharmacological properties of canine ERG potassium channels. Am J Physiol Heart Circ Physiol 2003, 284, (1), H256-67) have been shown to be predictive of hERG K+ channel block. However, the preparation, storage, and disposal of the radioligands adds time and cost to the assay procedure. Additionally, the radiometric assays that have been described to assess compound binding to the hERG K+ channel are heterogeneous filter binding assays, and require a separation of free from bound radioligand by capturing radioligand-bound membrane protein on filter paper using a vacuum manifold. This procedure makes the assay difficult to automate for large-scale screening or routine compound profiling, thereby limiting its practical utility. Additionally, over the past decade, there has been a strong push within both industry and academia to develop non-radioactive methods to replace such assays.
Fluorescence polarization (FP) assays provide a fully homogenous, mix-and-read format to characterize the affinity of a ligand for a receptor, and in many cases can be used to replace many radiometric binding assays (see, Burke, T. J.; Loniello, K. R.; Beebe, J. A.; Ervin, K. M., Development and application of fluorescence polarization assays in drug discovery. Comb Chem High Throughput Screen 2003, 6, (3), 183-94). The technique is based on the ability of a compound to displace a fluorescent probe (a “tracer”) from a receptor, which is detected by a change in an optical signal. In such an assay, the tracer typically consists of a known, high-affinity ligand for the receptor that has been chemically attached to a fluorescent molecule, without substantially disrupting the affinity of the receptor-ligand interaction (see, Huang, X., Fluorescence polarization competition assay: the range of resolvable inhibitor potency is limited by the affinity of the fluorescent ligand. J Biomol Screen 2003, 8, (1), 34-8). When a tracer molecule is excited with plane-polarized light in an FP assay, the polarization of the emitted light is retained if the fluorophore maintains its orientation during the time (typically nanoseconds) between photon excitation and emission. In solution, this orientation is largely maintained when the tracer is bound to a larger molecule, such as a protein, because the protein-tracer complex rotates more slowly than the free tracer itself. When the tracer is displaced from the receptor by a ligand that binds to the receptor, emission of light from the tracer is depolarized relative to the excitation source.
An important practical distinction between a traditional radioligand binding assay and an FP assay is that, in contrast to a radioligand binding assay, FP assays are optimally configured using a limiting amount of tracer, and a concentration of receptor that is at or above the Kd value for the receptor-tracer interaction. This is because the optical signal that is measured is dependant on the signal from all of the tracer that is present—both free and bound, which is unlike that in a radioligand binding assay in which (after separation) the only signal measured is due to bound ligand, and free ligand does not contribute to the signal. Thus, in an FP assay, any unbound tracer contributes to the amount of depolarized light present, thereby lowering the polarization signal that is measured, and lowering the assay window. Typically, FP assays are configured such that between 50 and 70% of the total tracer is bound in the absence of competing ligand in order to strike a balance between the assay window (maximal—minimal polarization values that are measured) and the assay sensitivity (ability of IC50 values to approach true Ki values) (see, Huang, X., Fluorescence polarization competition assay: the range of resolvable inhibitor potency is limited by the affinity of the fluorescent ligand. J Biomol Screen 2003, 8, (1), 34-8). When developing FP assays using purified, soluble, recombinant proteins, this is typically not an issue because many such proteins are readily prepared in quantities sufficient for such assays. However, this requirement can pose a challenge when developing assays for membrane-associated proteins, such as hERG, which in most cases have not been purified in functional form from their membrane components, which include both insoluble lipid components as well as other proteins. Moreover, the presence of large amounts of membrane components can interfere with the assay by scattering light (see, Banks, P.; Gosselin, M.; Prystay, L., Impact of a red-shifted dye label for high throughput fluorescence polarization assays of G protein-coupled receptors. J Biomol Screen 2000, 5, (5), 329-34) or by leading to increased non-specific binding of the tracer (which often contains a lipophilic fluorophore) with the membrane itself.
Accordingly, the development of a homogenous, FP-based assay to identify and characterize the affinity of small molecules for the hERG K+ channel, and demonstrate tight correlation with data obtained from either radioligand binding or patch-clamp assays, has heretofore not been realized.